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FITC is pH sensitive and not as bright or photostable as some of the newer fluorophores.  You might consider Alexafluor 488 or the Dylight488.  they can be conjugated to antibodies.  
A 2-step labelling method is usually brighter, using anti-sheep secondary antibody conjugated to the desired fluorophore. Keep the wash steps short ~5 min. if you are worried about antigen retention.
Sterility is not usually an issue except for either aliquot your immunoreagents or use a sterile pipettor tip to withdraw from the stock vial.
PBS with BSA for diluent.  Detergents are sometimes needed and sometimes folklore, best to test on your specimen with your antibody.  It may wash out the caffeine.
gelatin vs. bsa vs. blocking serum depends upon your materal and the antibody.  I've often used 0.1% gelatin, it stays liquid at 4 degrees C, but 1% will make jello salad.  You do need something in the diluent.  
Regards,
Glen

Glen MacDonald
Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
(206) 616-4156
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On Oct 13, 2010, at 9:08 AM, Shane Vontelin van Breda wrote:

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> *****
> 
> Unfortunately it wont be bound in any of the material. If the compound can
> with stand an entire embedding procedure I don't think using PBS etc will
> extract much.
> 
> We are doing a simulation of chemical fixation for the material and then we
> do an extraction and analyse it using HPLC. If caffeine is in the material
> there is a good chance it survives the embedding procedure. I will only know
> the results this Friday.
> 
> That is another reason why I would like to avoid as many wash steps as
> possible.
> 
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[log in to unmask]]On Behalf Of Russ Spear
> Sent: 13 October 2010 05:04 PM
> To: [log in to unmask]
> Subject: Re: Immunohistochemistry help needed.
> 
> 
> *****
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> 
> Hi
> 
> One concern I have, is it known if the caffeine is some how bound in
> this material, as caffeine is freely soluble in water in its pure form.
> 
> Other than that I normally use neat serum from the same species as the
> 2nd antibody as the blocking protein.
> 
> 
> Russ
> 
> On 10/13/2010 9:25 AM, Shane van Breda wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>> 
>> Dear list server members,
>> 
>> I am busy with my MSc and a while back I posted a question about
> localising
>> caffeine in tea (Camellia sinensis) leaves. We have made progress with
> normal
>> stains but I am ready to start using a confocal microscope and do some
>> immunohistochemistry work.
>> 
>> I have found three methods I would like to try. I would really appreciate
> it if I
>> could get some constructive criticsm on them.
>> 
>> Antibody: Sheep polyclonal anti – caffeine 5mg/ml.
>> 
>> Tracer: Fluorescein thiocarbamyl ethylendiamine (FTC-ED) (30nM/L working
>> concentration).
>> 
>> Diluent buffer: Na – phosphate buffer (100mM, pH 7.4) containing 1g/L
> bovine
>> gamma – globulins and 1g/L sodium azide. (Suppliers recommendation) or
> just
>> PBS and BSA is fine.
>> 
>> 1.)	Immunostaining of fixed samples:
>> 
>> Samples: Leaf blade containing caffeine, chemically fixed with
> formaldehyde,
>> dehydrated with EtOH and embedded in L.R. White or high pressure frozen
> and
>> freeze substituted in EtOH and formaldehyde and embedded in L.R. White or
>> high pressure frozen, freeze dried and embedded in L.R. White.
>> 
>> Procedure (direct labeling) obtained from light microscopy in biology
> edited by
>> A. J. Lacey:
>> 
>> 1.)	Make sections of +/- 1 – 5µm thick.
>> 2.)	Fix samples to slide using water or buffer (PBS) by applying heat
> (i.e.
>> evaporation). Or ‘free float samples’.
>> 3.)	Circle sections using a PAP pen.
>> 4.)	Place slide in humidity chamber (plastic Petri dish with water soaked
>> tissue and slide raised on a plastic bar).
>> 5.)	Place 0.5% (or 2%?) BSA , PBS on fixed sample for 10 - 15min.
>> 6.)	Rinse in PBS and wipe excess away.
>> 7.)	Place antibody on fixed samples in appropriated dilution i.e. 1:100.
>> Incubate for 1 hour at room temperature.
>> 8.)	Wash fixed sample in 0.5% (or 2%) BSA in PBS.
>> 9.)	Wash fixed sample in PBS, 0.01% detergent (Triton x100 or Tween
>> 80 in order to reduce the non-specific interaction between the Ig’s and
> the
>> proteins in the section.). Remove excess.
>> 10.)	Mark PAP pen marking with a black marker.
>> 11.)	Add a small amount of glycerol antifade mountant (n – propylgallate
>> or vectashield or 2mg/ml ascorbic acid etc) and add coverslip.
>> 
>> Questions:
>> 1.)	What would be a better buffer to use? PBS or sodium phosphate?
>> 2.)	Is it necessary to work in sterile conditions i.e. autoclave material
>> etc?
>> 3.)	Do you think there are unnecessary wash steps i.e. steps 6 and 8? I
>> am worried about washing out the antigen.
>> 
>> 2.) Immunostaining of fresh samples:
>> 
>> Sample: Fresh leaf containing caffeine.
>> 
>> Procedure: obtained from a PhD thesis titled Immunological localization of
> plant
>> secondary metabolites by Dr. Louise Brisson:
>> 
>> 1.)	Make hand sections with a sharp razor blade +/- 1mm thick.
>> 2.)	Adhere sample to slide using water or buffer (PBS) and mark with a
>> PAP pen or ‘free float samples’ and place slide in humidity chamber as
>> mentioned in section 1 (step 2 – 4).
>> 3.)	Block using 1% w/v gelatin in PBS (or osmoticum) for 15min.
>> 4.)	Incubate in antibody as mentioned in 1 (step 7).
>> 5.)	Rinse 3 times 3 min using PBS.
>> 6.)	Mount as in section 1 (step 10 and 11).
>> 
>> Questions:
>> 
>> 1.)	Should I include a step where the thick sections are incubated in
>> buffer and formaldehyde as a fixative?
>> 2.)	Instead of using gelatin in step 3 is it ok just to use BSA as in
>> section 1.
>> 3.)	Is it necessary to include a wash step before incubating with the
>> antibody i.e. just PBS?
>> 4.)	After incubation should I wash as in section 1 step 8 and 9 i.e. first
>> with PBS, BSA (or gelatin) then with PBS with detergent instead of 3 times
> 3
>> min using PBS?
>> 5.)	There is no step that includes the use of a detergent? Could this be
>> right? How could the antibody cross membranes then?
>> 6.)	Should I try this procedure as it is?
>> 
>> 
>> 3.) Immunostaining of cryofixed/cryoultramicrotome samples.
>> 
>> Sample: Fresh leaf containing caffeine.
>> 
>> Procedure: obtained from a PhD thesis titled Immunological localization of
> plant
>> secondary metabolites by Dr. Louise Brisson:
>> 
>> 1.)	Cut sections as in section 2 step 1.
>> 2.)	Embed in 20% w/v gelatin and rapidly freeze in N2 (l) and section
>> using a cryo ultramicrotome (+/- 8mm thick). Or use any type of cryo
> method
>> to achieve good sections.
>> 3.)	Labeling and fixing to a slide is done as steps 2 – 6 in section 2.
>> 
>> Questions:
>> 
>> 1.)	My questions are the same for section 2.
>> 2.)	Is it necessary to include a detergent since the sections are so thin?
>> Should the detergent only be included as a wash in order to reduce the
> non-
>> specific interaction between the Ig’s and the proteins in the section.
>> 
>> 
>> Thank you for reading my methods and any comments are welcomed.
>> 
>> Regards,
>> 
>> Shane Vontelin van Breda.
>> MSc. Biochemistry.
>> University of Pretoria.
>> Department of Biochemistry.
>> +27 83 998 6281.
>> [log in to unmask]
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